Method to inhibit cell growth using oligonucleotides

ABSTRACT

Described are methods for treating hyperproliferative disorders, including cancers, by administering to the affected mammal (e.g., human) an effective amount of a composition comprising one or more oligonucleotides which share at least 33% but less than 100% nucleotide sequence identity with the human telomere overhang repeat. Methods of treatment or prevention of hyperproliferative diseases or pre-cancerous conditions affecting epithelial cells, such as psoriasis, atopic dermatitis, or hyperproliferative diseases of other epithelia and methods for reducing photoaging, or oxidative stress or for prophylaxis against or reduction in the likelihood of the development of skin cancer, are also disclosed. The compositions and methods are also useful to treating other cancers.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/195,088 filed Aug. 1, 2005, which is a continuation-in-part of U.S.application Ser. No. 10/122,630 filed Apr. 12, 2002, now abandoned,which is a continuation-in-part of International Application No.PCT/US01/10162 filed on Mar. 30, 2001, which is a continuation-in-partof U.S. application Ser. No. 09/540,843 filed Mar. 31, 2000, now U.S.Pat. No. 7,094,766, which is a continuation-in-part of U.S. applicationSer. No. 08/952,697 filed Nov. 30, 1998, now abandoned, which is acontinuation-in-part of U.S. application Ser. No. 09/048,927 filed Mar.26, 1998, now U.S. Pat. No. 6,147,056, which is a continuation-in-partof International Application No. PCT/US96/08386 filed Jun. 3, 1996,which is a continuation-in-part of application Ser. No. 08/467,012 filedJun. 6, 1995, now U.S. Pat. No. 5,955,059. The entire teachings of theabove applications and issued patents are incorporated herein byreference.

BACKGROUND OF THE INVENTION

Mammalian cells have a complex response to DNA damage, as well as atightly regulated program of replicative senescence, all suggested to befundamental defenses against cancer [Campisi, J. (1996). Cell 84,497-500]. In mammals, cell senescence is precipitated by criticalshortening of telomeres, tandem repeats of the DNA sequence TTAGGG thatcap the ends of chromosomes [Greider, C. W. (1996) Annu Rev Biochem 65,337-365] and become shorter with each round of DNA replication. Ingermline cells and most cancer cells, immortality is associated withmaintenance of telomere length by telomerase, an enzyme complex thatadds TTAGGG repeats dues to the 3′ terminus at the chromosome ends[Feng, J., et al. Science 269, 1236-1241; Harrington, L., et al., (1997)Science 275, 973-977; Nakamura, T. M., et al., (1997) Science 277,955-957]. The catalytic subunit of telomerase is generally not expressedin normal somatic cells [Greider, C. W. (1996) Annu Rev Biochem 65,337-365], and after multiple rounds of cell division criticallyshortened telomeres trigger either replicative senescence or death byapoptosis, largely dependent on cell type [de Lange, T. (1998) Science279, 334-335], although the detailed mechanism is unknown. The mechanismby which telomeres participate in DNA damage responses has been lessclear.

The frequency of cancer in humans has increased in the developed worldas the population has aged. Melanoma and other skin cancers haveincreased greatly among aging populations with significant accumulatedexposure to sunlight. For some types of cancers and stages of disease atdiagnosis, morbidity and mortality rates have not improved significantlyin recent years in spite of extensive research. Cancers are currentlyoften treated with highly toxic therapies. Alternative therapies areneeded that could take advantage of the natural mechanisms of the cellsto repair environmental damage.

SUMMARY OF THE INVENTION

The present invention is directed to a composition comprising anoligonucleotide (T-oligo) having between 2 and 200 bases and having atleast 33% but less than 100% identity with the sequence (TTAGGG)_(n)where n=1 or greater and when said oligonucleotide comprises thesequence RRRGGG (R=any nucleotide) has a guanine content of 50% or less,said oligonucleotide optionally comprising an 5′ phosphate.

Preferred embodiments of the present invention comprise one or moresequences selected from the group consisting of TT, TA, TG, AG, GG, AT,GT, TTA, TAG, TAT, ATG, AGT, AGG, GAG, GGG, GGT, TTAG, TAG, AGGG, GGTT,GTTA, TTAGGG, TAGGG, GGTTA, GTTAG, GGGTT, and GGGGTT.

In still another preferred embodiment of the present invention, thecomposition is between about 40% and 90% identical to (TTAGGG)_(n). Inanother preferred embodiment, the compositions further comprise betweenabout 2 and 20 oligonucleotides or between 50 and 11 nucleotides.

Among the most preferred embodiments of the present invention is anoligonucleotide having a sequence selected from the group consisting of:

GGTTAGGGTGTAGGTTT; (SEQ ID NO: 28) GGTTGGTTGGTTGGTT; (SEQ ID NO: 29)GGTGGTGGTGGTGGT; (SEQ ID NO: 30) GGAGGAGGAGGAGGA; (SEQ ID NO: 31)GGTGTGGTGTGGTGT; (SEQ ID NO: 32) TAGTGTTAGGTGTAG; (SEQ ID NO: 34)GAGTATGAG; (SEQ ID NO: 1) AGTATGA; GTTAGGGTTAG; (SEQ ID NO: 2)GGTAGGTGTAGGATT; (SEQ ID NO: 10) GGTAGGTGTAGGTTA; (SEQ ID NO: 11)GGTTAGGTGTAGGTT; (SEQ ID NO: 12) GGTTAGGTGGAGGTTT; (SEQ ID NO: 13)GGTTAGGTTAGGTTA; (SEQ ID NO: 15) GGTTAGGTGTAGGTTT; (SEQ ID NO: 14)GTTAGGTTTAAGGTT; (SEQ ID NO: 19) and GTTAGGGTTAGGGTT. (SEQ ID NO: 22)

Another aspect of the invention encompasses the treatment ofhyperproliferative disorders comprising administering to a human acomposition comprising an oligonucleotide having between 2 and 200bases, and having at least 33% but less than 100% identity with thesequence (TTAGGG)_(n), where n=1 or greater, and when saidoligonucleotide comprises the sequence RRRGGG (R=any nucleotide) has aguanine content of 50% or less, the oligonucleotide optionallycomprising a 5′ phosphate and optionally lacks cytosine.

Another aspect of the present invention is a method promotingdifferentiation of malignant cells in a mammal, the method comprisingadministering to the mammal an effective amount of a compositioncomprising an oligonucleotide therebetween 2 and 200 bases and having atleast 33% but less than 100% identity with the sequence (TTAGGG)_(n),and when said oligonucleotide comprises the sequence RRRGGG (R=anyoligonucleotide) has a guanine content of 50% or less, saidoligonucleotide optionally comprising a 5′ phosphate and wherein saidoligonucleotide optionally lacks cytosine.

The method is further directed to a method for inducing apoptosis incancer cells in a human, the method comprising administering to thehuman an effective amount of a composition and comprising anoligonucleotide having between 2 and 200 bases and having at least 33%but less than 100% identity with the sequence (TTAGGG)_(n), where n=1 orgreater and when said oligonucleotide comprises the sequence RRRGGG(R=any oligonucleotide has a guanine content of 50% or less, and whereinsaid oligonucleotide optionally lacks cytosine.

Still another aspect of the present invention is a method for inhibitingthe growth of cancer cells in a human, the method being independent ofthe presence or activity of telomerase in the cancer cells, the methodcomprising the step of administering to a human in an effective amountof a composition and comprising an oligonucleotide having between 2 and200 bases and having at least 33% but less than 100% identity with thesequence (TTAGGG)_(n), when said oligonucleotide comprises the sequenceRRRGGG (R=any oligonucleotide has a guanine content of 50% or less.

A still further aspect of the invention is a method to inhibit thegrowth of cancer cells in a human. The method not requiring the presenceor activity of p53 gene product in cancer cells, the method comprisingadministering a composition comprising an oligonucleotide having between2 and 200 bases and having at least 33% but less than 100% identity withthe sequence (TTAGGG)_(n), where n=1 or greater and when saidoligonucleotide comprises the sequence RRRGGG (R=any oligonucleotide)has a guanine content of 50% or less.

A still further aspect of the present invention is a method to inhibitthe growth of cancer cells in a human, the method resulting in anS-phase arrest in said cells, the method comprising administering to thehuman an effective amount of a composition and comprising anoligonucleotide having between 2 and 200 bases and having at least 33%but less than 100% identity with the sequence (TTAGGG)_(n), where n=1 orgreater and when said oligonucleotide comprises the sequence RRRGGG(R=any oligonucleotide) has a guanine content of 50% or less.

The invention is also directed to a method for preventing spongiosis,blistering, or dyskeratosis in the skin of a mammal following exposureto ultraviolet light, the method comprising apply to the skin aneffective amount of a composition comprising an oligonucleotide havingbetween 2 and 200 bases and having at least 33% but less than 100%identity with the sequence (TTAGGG)_(n), where n=1 or greater and whensaid oligonucleotide comprises the sequence RRRGGG (R=anyoligonucleotide) has a guanine content of 50% or less.

Also encompassed by the invention is a method for reducing theoccurrences of skin cancer in a human, the method comprising applying tothe skin an effective amount of a composition comprising anoligonucleotide having between 2 and 200 bases and having at least 33%but less than 100% identity with the sequence (TTAGGG)_(n), where n=1 orgreater and when said oligonucleotide comprises the sequence RRRGGG(R=any oligonucleotide) has a guanine content of 50% or less.

Another embodiment of the present invention is directed to methods toreduce the occurrence of skin cancer in a human with xerodermapigmentosum or other genetically determined cancer predisposition, themethod comprising applying to the skin, an effective amount of acomposition comprising an oligonucleotide having between 2 and 200 basesand having at least 33% but less than 100% identity with the sequence(TTAGGG)_(n), where n=1 or greater and cytosine and when saidoligonucleotide comprises the sequence RRRGGG (R=any oligonucleotide)has a guanine content of 50% or less.

Also included in the present invention is a method for enhancing repairof ultraviolet irradiation induced damage to skin in a human in whichthe method includes applying to the skin an effective amount of acomposition comprising an oligonucleotide having between 2 and 200 basesand having at least 33% but less than 100% identity with the sequence(TTAGGG)_(n), where n=1 or greater and when said oligonucleotidecomprises the sequence RRRGGG (R=any oligonucleotide) has a guaninecontent of 50% or less.

The invention is also directed to a method for reducing oxidative damagein a mammal. The method comprising administering to the mammal aneffective amount of a composition comprising an oligonucleotide havingbetween 2 and 200 bases and having at least 33% but less than 100%identity with the sequence (TTAGGG)_(n), where n=1 or greater and whensaid oligonucleotide comprises the sequence RRRGGG (R=anyoligonucleotide) has a guanine content of 50% or less.

The present invention is also directed to a method for reducingproliferation of keratinocytes in the skin of a human. The methodcomplying applying to the skin a composition comprising anoligonucleotide having between 2 and 200 bases and having at least 33%but less than 100% identity with the sequence (TTAGGG)_(n), where n=1 orgreater and when said oligonucleotide comprises the sequence RRRGGG(R=any oligonucleotide) has a guanine content of 50% or less.

Still another embodiment comprises increasing DNA repair in cells andpreferably in epithelial cells comprising contacting the epithelialcells with an effective amount of a composition comprising anoligonucleotide having between 2 and 200 bases and having at least 33%but less than 100% identity with the sequence (TTAGGG)_(n), where n=1 orgreater and when said oligonucleotide comprises the sequence RRRGGG(R=any oligonucleotide) has a guanine content of 50% or less.

Other oligonucleotides useful in the practice of any of the methods ofthe present invention are known as G-quadruplex DNAs, or alternativelyG-tetraplex DNAs.

Preferred G-quadruplex DNAs useful in the methods of the presentinvention comprise from about 3 nucleotides to about 200 nucleotides.Preferably the G-quadruplex DNA or RNA is at least 33% identical to(TTAGGG)_(n) where n=1 or greater and preferably is less than 100%identical to (TTAGGG)_(n), where n=1 or greater. More preferably, theG-quadruplex DNA is between 40% and 60% identical to (TTAGGG)_(n).Preferably, the G-quadruplex DNA lacks cytosine. Preferably, when theG-quadruplex DNA comprises the sequence RRRGGG (R=any nucleotide), ithas a guanine content of less than 50% and wherein said G-quadruplex DNAoptionally comprises cytosine and preferably the G-quadruplex DNA has a5′ and/or 3′ single strand.

A further method, useful in the treatment of cancers, is a method forenhancing the expression of one or more surface antigens indicative ofdifferentiation of cancer cells in a human, the method comprisingadministering to the human an effective amount of an oligonucleotide ofthe present invention. The cells in this method can be, for example,melanoma, and the antigen can be, for example, MART-1, tyrosinase, TRP-1or gp-100. The cells can, for example, be breast cancer cells and theantigen can be estrogen receptor α. Further, the invention is a methodfor inducing apoptosis in cancer cells in a human, said methodcomprising administering to the human an effective amount of acomposition comprising one or more oligonucleotides which share at least50% nucleotide sequence identity with the human telomere overhangrepeat. This method can be applied, for example, to melanoma or to anyother malignancy.

Thus, another method of the invention is a method for inducingsenescence in cancer cells in a mammal (e.g., a human), the methodcomprising administering to the mammal an effective amount of acomposition comprising one or more oligonucleotides encompassed by thepresent invention.

Also a part of the invention is a method for inhibiting the growth ofcancer cells in a human, the method being independent of the presence oractivity of telomerase in the cancer cells, in which the method includesthe step of administering to the human an effective amount of acomposition comprising one or more oligonucleotides encompassed by thepresent invention.

A further aspect of the invention is a method to inhibit the growth ofcancer cells in a human, the method not requiring the presence oractivity of p53 gene product in the cancer cells, the method comprisingadministering to the human an effective amount of a compositioncomprising one or more oligonucleotides as described above.

A further aspect of the invention is a method to inhibit the growth ofcancer cells in a human, the method resulting in S-phase arrest in saidcells, the method comprising administering to the human an effectiveamount of a composition comprising one or more oligonucleotides. Theoligonucleotide to be used can be various lengths, but in one embodimentthe oligonucleotide can be less than 6 nucleotides long.

The present invention is also directed to a method for preventingspongiosis, blistering or dyskeratosis in the skin of a mammal,following exposure to ultraviolet light, the method comprising applyingto the skin an effective amount of a composition comprising one or moreoligonucleotides.

Still another aspect of the invention is a method for reducing theoccurrence of skin cancer in a human, the method comprising applying tothe skin an effective amount of a composition comprising one or moreoligonucleotides.

In another aspect of the methods of the present invention to reduce theoccurrence of skin cancer in a human is a method for reducing theoccurrence of skin cancer in a human with xeroderma pigmentosum, orother genetically determined cancer predisposition, the methodcomprising applying to the skin an effective amount of a compositioncomprising one or more oligonucleotides according to the presentinvention.

Also included in the invention is a method for enhancing repair ofultraviolet irradiation-induced damage to skin in a human, in which themethod includes applying to the skin an effective amount of acomposition comprising one or more oligonucleotides of the presentinvention.

Also included as an aspect of the invention is a method for reducingoxidative damage in a mammal, the method comprising administering to themammal an effective amount of a composition comprising one or moreoligonucleotides of the present invention.

It is also an object of the invention to provide a method for treatingmelanoma in a mammal, comprising administering to the mammal aneffective amount of a composition comprising one or moreoligonucleotides of the present invention. Various combinations of theseoligonucleotides can also be used in the method.

It is also an object of the invention to provide a method for reducingproliferation of keratinocytes in the skin of a human, the methodcomprising applying to the skin an effective amount of a compositioncomprising one or more oligonucleotides according to the presentinvention. In particular applications of the method, the human to betreated has seborrheic keratosis, actinic keratosis, Bowen's disease,squamous cell carcinoma, or basal cell carcinoma. Another embodimentcomprises increasing DNA repair in epithelial cells, comprisingcontacting said cells with an effective amount of a compositioncomprising at least one oligonucleotide, and a contiguous portion of anyof the foregoing sequences. Preferred oligonucleotides for use in themethods of the present invention include but are not limited to SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ IDNO:35, SEQ ID NO:36, and a fragment of any of the foregoing sequences,preferably including from about two nucleotides, more preferably 3 ormore contiguous nucleotides of the full length oligonucleotide.

Also included in the invention are truncated versions of the aboveoligonucleotides identified above. The oligonucleotides of the presentinvention may be truncated by one or more nucleotides on the 5′ end, the3′ end, or both the 5′ end and the 3′ end, so long as at least twocontiguous nucleotides of the untruncated oligonucleotide remain.Preferably the truncated oligonucleotides have 10, 9, 8, 7, 6, 5, 4, 3or 2 contiguous nucleotides found in the untruncated nucleotides.

Also a part of the invention are compositions comprising one or moreoligonucleotides in a physiologically acceptable carrier, wherein theoligonucleotide comprises base sequence SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17. Further, the invention can be a compositioncomprising one or more oligonucleotides in a physiologically acceptablecarrier, wherein the oligonucleotide consists of base sequence SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ IDNO:35, or SEQ ID NO:36.

Preferred for applications in which it is desired to inhibit cellproliferation or to induce apoptosis are oligonucleotides comprising SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:35, or SEQ ID NO:36.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—Induction of apoptosis in MM-AN cells by a 16 mer and 20 meroligonucleotide.

FIG. 2—Induction of apoptosis in MM-AN cells by variousoligonucleotides.

FIG. 3—Induction of phosphorylation of H2AX by various oligonucleotides.

FIG. 4—Induction of phosphorylation of H2AX in normal mammary epithelialcells and MCF-7 breast tumor cells.

FIG. 5—Induction of phosphorylation of p53 in normal mammary epithelialcells and MCF-7 breast tumor cells

FIG. 6—Effect of oligonucleotides on growth of normal mammary epithelialcells.

FIG. 7—Effect of oligonucleotides on growth of MCF-7 breast tumor cells.

FIG. 8—Effect of oligonucleotides on growth of BT20 breast tumor cells.

FIG. 9—Effect of oligonucleotides on survival of mice injected withMCF-7 breast cancer cells.

FIG. 10—Effect of intratumoral injection oligonucleotides on squamouscell carcinoma in mice.

FIG. 11—Effect of oligonucleotides on MM-AN melanoma in SCID mice.

FIG. 12—Effect of oligonucleotides on weight loss in SCID mice withmelanoma.

FIG. 13—Effect of oligonucleotides on average tumor volume in SCID micewith MM-AN melanoma.

FIG. 14—Effect of oligonucleotides on average metastasis number in SCIDmice with MM-AN melanoma.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that treatment of cellswith oligonucleotides having at least 33% but less than 100% identitywith the sequence (TTAGGG)_(n) can elicit a variety of responsesincluding inhibition of cell proliferation, apoptosis, induction of DNArepair a protective response to exposure to U.V.-irradiation, otherionizing radiation or carcinogenic chemicals and other results.

More specifically, the invention pertains to the use of sucholigonucleotides a similar compounds, for the inhibition of cellproliferation the induction of senesence, or induction of apoptosis orinduction of DNA repair. As used herein, inhibition of cellproliferation includes complete abrogation of cell division, partialinhibition of cell division and transient inhibition of cell division asmeasured by standard tests in the art and as described in the Examplesherein and in PCT/US03/11393 which is incorporated herein by references.The invention also pertains to the prevention and/or treatment ofhyperproliferative diseases, using the oligonucleotides of the presentinvention including, the diseases including but not limited to, cancerand pre-cancerous conditions, wherein the hyperproliferative diseaseaffects cells of any organ and are of any embryonic origin. Tumorsincluding metastatic tumors and cancers that have regrown or relapsedafter treatment, as well as primary tumors, can be treated by themethods and materials of the invention. In particular embodiments, thediseases and conditions to be treated include skin diseases such aspsoriasis and hyperproliferative, pre-cancerous or U.V.-induceddermatoses in mammals, particularly in humans as well as a variety oftumors including melanoma, breast cancer, prostate cancer, as well as avariety of cancer hematological malignancies, lung cancer, and othercarcinomas.

The invention further pertains to use of the oligonucleotides of thepresent invention to reduce photoaging (a process due in part tocumulative DNA damage), and to reduce oxidative stress and oxidativedamage. The invention also pertains to prophylaxis against, or reductionin the likelihood of, the development of skin cancer in a mammal usingthe oligonucleotides of the present invention. In addition, thecompounds of the present invention can be used to induce apoptosis incells such as cells that have sustained genetic mutation, such asmalignant or cancer cells or cells from an actinic keratosis.

All types of cells, and in particular embodiments, epithelial cells, areexpected to respond to the methods of the present invention asdemonstrated by the representative in vitro and in vivo examplesprovided herein. Epithelial cells suitable for the method of the presentinvention include epidermal cells, respiratory epithelial cells, nasalepithelial cells, oral cavity cells, aural epithelial cells, ocularepithelial cells, genitourinary tract cells and esophageal cells, forexample. Gastrointestinal cells are also contemplated in methods of theinvention as described herein.

Cells that contain damaged or mutated DNA include, for example, actinickeratosis cells, cancer cells, cells that have been irradiated, as withU.V. light, and cells that have been exposed to DNA damaging chemicalsor conditions will also respond to the oligonucleotides of the presentinvention. Inflammation, including allergically mediated inflammationinvolved in conditions such as atopic dermatitis, contact dermatitis,allergic rhinitis and allergic conjunctivitis may also be treated usingthe oligonucleotides of the present invention.

In one embodiment, the compositions of the present invention compriseDNA oligonucleotides approximately 2-200 bases in length, having atleast 33% but less than 100% identity with the sequence (TTAGGG)_(n)where n=1 or greater and optionally having a 5′ phosphate and when saidoligonucleotide comprises the sequence 5′ RRRGGG-3′ (R=any nucleotide)the oligonucleotide has a guanine content of 50% or less which can beadministered to a mammal (e.g., human) in an appropriate vehicle. Inanother embodiment, the DNA oligonucleotides are about 2 to about 20nucleotides in length. In still another embodiment, the oligonucleotidesare about 5 to about 11 nucleotides in length. In yet anotherembodiment, the DNA oligonucleotides are about 2-5 nucleotides inlength. Certain preferred embodiments of the oligonucleotides of thepresent invention lack cytosine. As used herein, “DNA oligonucleotide”refers to single-stranded DNA oligonucleotides, double-stranded DNAfragments, or a mixture of both single- and double-stranded DNAfragments.

It is understood that other base-containing sequences can also be usedin the present invention, where bases are, for example, adenine,thymine, cytosine, or guanine. In one embodiment, the oligonucleotidesof the present invention comprise a 5′ phosphate. A combination of oneor more of oligonucleotides of the present invention can also be used.

Other compositions useful in the practice of the present inventioninclude G-quadruplex DNA, also known as DNA tetraplexes. Guanine basesin solution can form a structure consisting of four bases in a planararray and held together by Hoogsteen hydrogen bonds, calledG-quadruplexes or tetraplexes. Similarly, poly(G) homopolymers also form4-stranded nucleic acid structures with stacked guanine tetrads. In DNA,guanine tetraplexes can form from intrastrand or interstrandassociations with 2 or more of the tetrads stacked upon each other tostabilize the complex. These strands can run in a parallel or ananti-parallel fashion with non G-rich DNA looped out and connecting thetetrad cores.

For many years, G-quadruplexes were considered to be an interesting butnon-biologically relevant phenomenon. However, recently G-quadruplexeshave been suggested to be important in immunoglobulin heavy chainrecombination and in the regulation of the retinoblastoma and c-mycgenes. Also, G-quadruplexes have been shown to form in single-strandedtelomeric DNA, particularly in the 3′ overhang. Although the function ofthese G-quadruplexes in telomeres, if any, is unclear, it has beenpostulated that they may play a role in protein recognition and telomererecombination. More recently, G-quadruplex formation in telomeric DNAwas shown to inhibit telomere elongation by telomerase and ligands thatstabilize G-quadruplexes are now in trials as telomerase inhibitors andanticancer agents.

Theoretically, G-quadruplexes could stall the procession of DNA and RNApolymerases, hindering DNA replication and translation, situations thatcould lead to increased DNA recombination and mutation. Two DNAhelicases have been identified that have a high affinity for theseunusual DNA structures and can resolve them efficiently; Wrn and Blm,the helicases mutated in the diseases Werner Syndrome and BloomSyndrome, respectively. Wrn is of particular interest because itcontains both 3′ to 5′ helicase and exonuclease activities and is ableto catalyze the structure-dependent degradation of DNA containingaberrant structures such as bubbles, loops and hairpins and of G-richtelomeric DNA specifically. Furthermore, cells from Werner Syndromepatients shows signs of impaired telomere maintenance such asaccelerated telomere shortening and defective lagging strand synthesis(20). Because of Wrn's preference for unusual DNA structures, it ispossible that Wrn-mediated telomere functions depend, at least in part,on the ability of single-stranded telomeric DNA, particularly the 3′overhang, to form G-quadruplex structures.

We have shown that small DNA oligonucleotides homologous to the telomere3′ overhang (T-oligos) induce DNA damage responses in normal andtransformed mammalian cells and that these responses are largelydependent on Wrn and the degradation of these oligonucleotides. The beststudied of these oligonucleotides to date is the 11mer GTTAGGGTTAG (SEQID NO:5). It is possible that Wrn-mediated degradation of theseoligonucleotides initiates these DNA damage responses. However, we havefound that oligonucleotides other than those 100% identical to thetelomere repeat sequence of (TTAGGG)_(n) are also very potent inducersof these DNA damage responses as described herein. For example, the16-mer GGTTGGTTGGTTGGTT (SEQ ID NO:29) and the 20-merGGTTGGTTGGTTGGTTGGTT (SEQ ID NO:35) are the most potent inducers of theDNA damage response found to date. Because of the ability of theseT-oligos to form an intrastrand G-quartet stabilized by 2 overlappingplanar G-tetrads (the interstrand G-quartet formed by GTTAGGGTTAG (SEQID NO:5) would be less stable), we believe that the activity of T-oligosdepends at least in part on their ability to form these stable,intrastrand G-quartets. These structures would be excellent substratesfor the Wrn helicases/exonuclease and, accordingly, are likely to be themost active inducers of the DNA damage responses.

PCT/US03/11393

PCT/US03/11393 from which the present application claims prioritydescribes the use of a nine-nucleotide oligomer, GAGTATGAG (SEQ ID NO:1)that stimulates melanogenesis in human melanocytes and induced theexpression of p21/Waf/Cip 1, a growth inhibitory gene product in asquamous cell carcinoma cell line. Furthermore, TAGGAGGAT (SEQ ID NO:40)(5/9 identity with telomere overhang repeat), and truncated versions ofthe original 9mer, AGTATGA and GTATG, also stimulated melanogenesis inhuman melanocytes. In addition, the sequence pGTTAGGGTTAG (SEQ ID NO:5)stimulated pigmentation in Cloudman S91 melanoma cells and inducedapoptosis in a human T-cell line. The oligonucleotide pGTTAGGGTTAG (SEQID NO:5) induced human T cells to undergo apoptosis, while CTAACCCTAAC(SEQ ID NO:3) and pGATCGATCGAT (SEQ ID NO:39) did not significantlyincrease apoptosis in these cells.

The compounds of the present invention are therefore useful in methodsof inhibiting cell proliferation, preventing cancer, photoaging andoxidative stress by enhancing DNA repair, and, in the skin, by enhancingpigmentation through increased melanin production. Melanin is known toabsorb photons in the U.V. range and therefore its presence reduces therisk of cancer and photoaging.

The DNA fragments, oligonucleotides, deoxynucleotides, dinucleotides,and dinucleotide dimers can be obtained from any appropriate source, orcan be synthetically produced. To make DNA fragments, for example,salmon sperm DNA can be dissolved in water, and then the mixture can beautoclaved to fragment the DNA. In one embodiment, the DNA fragments,oligonucleotides, deoxynucleotides, dinucleotides or dinucleotide dimerscomprise a 5′ phosphate.

The compounds of the present invention also play a protective role inU.V.A-induced oxidative damage to the cell (see e.g. Example 15 ofPCT/US03/11393). Thus, in one embodiment of the present invention, thecompounds of the present invention are administered to cells to protectagainst oxidative damage. In one embodiment, these compounds aretopically administered to the epidermis of an individual.

An “agent that increases activity of p53 protein,” as used herein, is anagent (e.g., a drug, molecule, nucleic acid fragment, oligonucleotide,or nucleotide) that increases the activity of p53 protein and thereforeresults in increase in an DNA repair mechanisms, such as nucleotideexcision repair, by the induction of proteins involved in DNA repair,such as PCNA, XPB and p21 proteins. The activity of p53 protein can beincreased by directly stimulating transcription of p53-encoding DNA ortranslation of p53-specific mRNA, by increasing expression or productionof p53 protein, by increasing the stability of p53 protein, byincreasing the resistance of p53 mRNA or protein to degradation, bycausing p53 to accumulate in the nucleus of a cell, by increasing theamount of p53 present, by phosphorylating the serine 15 residue in p53,or by otherwise enhancing the activity of p53. A combination of morethan one agent that increases the activity of p53 can be used.Alternatively or in addition, the agent that increases the activity ofp53 can be used in combination with DNA fragments, deoxynucleotides, ordinucleotides, as described above.

Ultraviolet irradiation produces DNA photoproducts that when notpromptly removed, can cause mutations and skin cancer. Repair ofU.V.-induced DNA damage requires efficient removal of the photoproductsto avoid errors during DNA replication. Age-associated decrease in DNArepair capacity is associated with decreased constitutive levels of p53and other nuclear excision repair (NER) proteins required for removingU.V.-induced photoproducts. Compounds of the present invention inducedNER proteins in human dermal cells when these cells were treated withthese compounds before U.V. irradiation (see Example 16 ofPCT/US03/11393). While there were age related decreases in NER proteins,NER proteins in cells from donors of all ages from newborn to 90 yearswere induced by 200-400%. A significant decrease in the rate of repairof thymine dimers and photoproducts occurs with increased age of thecell sample; however, cells that were pre-treated with compounds of thepresent invention, then U.V. irradiated, removed photoproducts 30 to 60percent more efficiently. Thus, the treatment of cells with small DNAoligonucleotides partially compensates for age-associated decreases inDNA repair capacity. In light of the in vivo efficacy of the compoundsof the present invention, it is reasonable to expect that treatment ofhuman skin with the compounds of the present invention enhancesendogenous DNA repair capacity and reduces the carcinogenic risk fromsolar U.V. irradiation. This method is especially useful in olderindividuals who likely have reduced cellular DNA repair capacity.

DNA fragments, oligonucleotides, deoxynucleotides, dinucleotides ordinucleotide dimers, to be applied to the skin in methods to prevent thesequelae of U.V. exposure or to reduce the occurrence of skin cancer, toreduce oxidative damage, or to enhance repair of U.V.-induced damage,can be administered alone, or in combination with physiologicallyacceptable carriers, including solvents, perfumes or colorants,stabilizers, sunscreens or other ingredients, for medical or cosmeticuse. They can be administered in a vehicle, such as water, saline, or inanother appropriate delivery vehicle. The delivery vehicle can be anyappropriate vehicle which delivers the DNA fragments, oligonucleotides,deoxynucleotides, dinucleotides, or dinucleotide dimers. In oneembodiment, the concentration of oligonucleotide can be 10 μM-100 μM.

To allow access of the active ingredients of the composition todeeperlying skin cells, vehicles which improve penetration through outerlayers of the skin, e.g., the stratum corneum, are useful. Vehicleconstituents for this purpose include, but are not limited to, ethanol,isopropanol, diethylene glycol ethers such as diethylene glycolmonoethyl ether, azone (1-dodecylazacycloheptan-2-one), oleic acid,linoleic acid, propylene glycol, hypertonic concentrations of glycerol,lactic acid, glycolic acid, citric acid, and malic acid. In oneembodiment, propylene glycol is used as a delivery vehicle. In apreferred embodiment, a mixture of propylene glycol:ethanol:isopropylmyristate (1:2.7:1) containing 3% benzylsulfonic acid and 5% oleylalcohol is used.

In another embodiment, a liposome preparation can be used. The liposomepreparation can comprise liposomes which penetrate the cells of interestor the stratum corneum, and fuse with the cell membrane, resulting indelivery of the contents of the liposome into the cell. For example,liposomes such as those described in U.S. Pat. No. 5,077,211 of Yarosh,U.S. Pat. No. 4,621,023 of Redziniak et al. or U.S. Pat. No. 4,508,703of Redziniak et al., all of which are incorporated herein by referencecan be used. The compositions of the invention intended to target skinconditions can be administered before, during, or after exposure of theskin of the mammal to U.V. or agents causing oxidative damage. Othersuitable formulations can employ niosomes. Niosomes are lipid vesiclessimilar to liposomes, with membranes consisting largely of non-ioniclipids, some forms of which are effective for transporting compoundsacross the stratum corneum.

Other suitable delivery methods intended primarily for skin include useof a hydrogel formulation, comprising an aqueous or aqueous-alcoholicmedium and a gelling agent in addition to the oligonucleotide(s).Suitable gelling agents include methylcellulose, carboxymethylcellulose,hydroxypropylmethylcellulose, carbomer (carbopol), hypan, polyacrylate,and glycerol polyacrylate.

In one embodiment, oligonucleotides, or composition comprising one ormore of the foregoing, is applied topically to the skin surface. Inother embodiments, the DNA fragments, oligonucleotides,deoxynucleotides, dinucleotides, dinucleotide dimers, or compositioncomprising one or more of the foregoing, is delivered to other cells ortissues of the body such as epithelial cells. Cells of tissue that isrecognized to have a lesser barrier to entry of such substances thandoes the skin can be treated, e.g., orally to the oral cavity; byaerosol to the respiratory epithelium; by instillation to the bladderepithelium; by instillation or suppository to intestinal (epithelium) orby other topical or surface application means to other cells or tissuesin the body, including eye drops, nose drops and application usingangioplasty, for example. Furthermore, the oligonucleotides of thepresent invention can be administered intravenously or injected directlyinto the tissue of interest intracutaneously, subcutaneously,intramuscularly or intraperitoneally, along with a pharmaceuticallyacceptable carrier. In addition, for the treatment of blood cells, thecompounds of the present invention can be administered intravenously orduring extracorporeal circulation of the cells, such as through aphotophoresis device, for example. As demonstrated herein, all that isneeded is contacting the cells of interest with the oligonucleotidecompositions of the present invention, wherein the oligonucleotidescontacting the cells can be as small as dinucleotides.

The oligonucleotides, deoxynucleotides, dinucleotides, dinucleotidedimers, agent that promotes differentiation, agent that increases p53activity, or composition comprising one or more of the foregoing, isadministered to (introduced into or contacted with) the cells ofinterest in an appropriate manner. The “cells of interest,” as usedherein, are any cells which may become affected or are affected by thehyperproliferative disease precancerous condition or cancerousconditions, or cells which are affected by oxidative stress, DNAdamaging conditions such as U.V. irradiation or exposure to damaging DNAchemicals such as benzo[a]pyrene. Preferred cells are epithelial cells,including melanocytes and keratinocytes, as well as other epithelialcells such as oral, respiratory, bladder and cervical epithelial cells.As demonstrated herein, methods and compositions of the presentinvention can inhibit growth, induce melanogenesis and induce TNFaproduction in epithelial cells from numerous sources.

The oligonucleotides, DNA fragments, deoxynucleotides, dinucleotides,dinucleotide dimers, agent that promotes differentiation, that increasesp53 activity, or compositions comprising one or more of the foregoing,is applied at an appropriate time, in an effective amount. The“appropriate time” will vary, depending on the type and molecular weightof the oligonucleotides, DNA fragments, deoxynucleotides, dinucleotides,dinucleotide dimers, or other agent employed, the condition to betreated or prevented, the results sought, and the individual patient. An“effective amount,” as used herein, is a quantity or concentrationsufficient to achieve a measurable desired result. The effective amountwill depend on the type and molecular weight of the oligonucleotides,DNA fragments, deoxynucleotides, dinucleotides, dinucleotide dimers, oragent employed, the condition to be treated or prevented, the resultssought, and the individual patient. For example, for the treatment orprevention of psoriasis, or for hyperproliferative, cancerous, orpre-cancerous conditions, or U.V.-induced dermatoses, the effectiveamount is the amount necessary to reduce or relieve any one of thesymptoms of the disease, to reduce the volume, area or number of cellsaffected by the disease, to prevent the formation of affected areas, orto reduce the rate of growth of the cells affected by ahyperproliferative disorder. The concentration can be approximately2-300 μM. In a another embodiment, the concentration of agent (e.g.,oligonucleotide) is about 50-200 μM; in another embodiment, theconcentration is about 75-150 μM. For some applications, theconcentration of oligonucleotide can be about 10-100 μM.

In one embodiment of the present invention, oligonucleotides, agent thatincreases p53 activity, that promotes differentiation, or a compositionthat can comprise one or more of the foregoing, is administered, in anappropriate delivery vehicle, to the cells of interest in the mammal inorder to treat or prevent a hyperproliferative disease affectingepithelial cells. The oligonucleotides, that promote differentiation,agent that increases p53 activity, or composition comprising one or moreof the foregoing, can be administered systemically, can be administereddirectly to affected areas, or can be applied prophylactically toregions commonly affected by the hyperproliferative disease.

In another embodiment, the DNA fragments, oligonucleotides,deoxynucleotides, dinucleotides, dinucleotide dimers, agent thatincreases p53 activity, agent that promotes differentiation, orcomposition comprising one or more of the foregoing, is administered tothe epidermis for the treatment or prevention of oxidative stress or forthe treatment or prevention of hyperproliferative, cancerous, orpre-cancerous conditions, or LJV-responsive dermatoses.

In still another embodiment, DNA fragments, oligonucleotides,deoxynucleotides, dinucleotides, dinucleotide dimers, agent thatpromotes differentiation, agent that increases p53 activity, or acomposition comprising one or more of the foregoing, can beadministered, either alone or in an appropriate delivery vehicle, to theepidermis for reduction of photoaging, or prophylaxis against orreduction in the likelihood of development of skin cancer. The DNAfragments, oligonucleotides, deoxynucleotides, dinucleotides,dinucleotide dimers, agent that promotes differentiation, agent thatincreases p53 activity, or composition comprising one or more of theforegoing, can be administered topically or by intracutaneous injectionat an appropriate time (i.e., prior to exposure of the skin to U.V.irradiation). The DNA fragments, oligonucleotides, deoxynucleotides,dinucleotides, dinucleotide dimers, agent that promotes differentiation,agent that increases p53 activity, or composition comprising one or moreof the foregoing, can be applied before, during or after exposure to acarcinogen such as U.V. irradiation. They can be applied daily or atregular or intermittent intervals. In one embodiment, the DNA fragments,oligonucleotides, deoxynucleotides, dinucleotides, dinucleotide dimers,agent that promotes differentiation, agent that increases p53 activity,or composition comprising one or more of the foregoing, can beadministered on a daily basis to skin which may be exposed to sunlightduring the course of the day.

In a further embodiment of the invention, the DNA fragments,oligonucleotides, deoxynucleotides, dinucleotides, dinucleotide dimers,agent that promotes differentiation, agent that increases p53 activity,or composition comprising one or more of the foregoing, is administered,in an appropriate delivery vehicle, to an individual (e.g., epithelialcells or other cells of an individual) for the treatment or preventionof hyperproliferative, cancerous or pre-cancerous conditions, or torepair or prevent DNA damage caused by DNA damaging chemicals, such asbenzo[a]pyrene.

As demonstrated herein, the compounds of the present invention areactive in vitro and in vivo in their unmodified form, e.g., sequences ofunmodified oligonucleotides linked by phosphodiester bonds. As usedherein, the terms “oligonucleotide,” “dinucleotide,” “DNA fragment,”etc., refer to molecules having deoxyribose as the sugar, and havingphosphodiester linkages (“phosphate backbone”) as occur naturally,unless a different linkage or backbone is specified.

Furthermore, although not necessary for the ability to elicit the U.V.mimetic effects of the present invention, the compounds of the presentinvention can be modified, derivative or otherwise combined with otherreagents to increase the half life of the compound in the organismand/or increase the uptake of these compounds by the cells of interest.Modification reagents include, for example, lipids or cationic lipids.In one embodiment, the compounds of the present invention are covalentlymodified with a lipophilic group, an adamancy moiety. The compounds ofthe present invention can be modified to target specific tissues in thebody. For example, brain tissue can be targeted by conjugating thecompounds with biotin and using the conjugated compounds with an agentthat facilitates delivery across the blood-brain barrier, such asantitransferring receptor antibody coupled to streptavidin.

PCT/US03/11393 demonstrates that telomere homolog oligonucleotides butnot complementary or unrelated DNA sequences of the same length inducean S-phase arrest and apoptosis in an established human lymphocyte lineand in a human melanoma cell line.

Experimental telomere disruption [Karlseder, J., et al., 1999, Science283: 1321-1325] and cellular manipulations that precipitate prematuresenescence, such as transfection with the ras oncogene or exposure tooxidative stress, are known to digest the 3′ telomere overhang and/or toshorten overall telomere length. In contrast, exposure of cells totelomere homolog oligonucleotides in the present studies increases meantelomere length (MTL). While not wishing to be bound by a singlemechanism, these data strongly suggest that the oligonucleotides canactivate telomerase, presumably by inducing TERT, and imply thattransient activation of telomerase may be a part of the physiologictelomere-based DNA damage response that also includes activation of theATM kinase with subsequent signaling through p53 and p95/Nbs 1. Theapparent ability of oligonucleotides to induce this response in theabsence of DNA damage and telomere disruption offers the possibility of“rejU.V.enating” cells through telomere elongation, as recently reportedin human skin equivalent constructs containing fibroblasts transfectedwith TERT, without the enhanced risk of carcinogenesis observed even ineven normal cells that ectopically express telomerase. Equally, thephenomenon suggests that the advantages of robust DNA damage responsesof the type observed in p53 overexpressing mice could be separated fromthe premature senescence also observed in the transgenic animals (Tyner,S. D., et al., 2002, Nature 415: 45-523). Such “rejuvenation” or delayin acquiring the senescent phenotype associated with critical telomereshortening would be in addition to other benefits that might accrue fromtreatment with oligonucleotides partially or completely homologous tothe (TTAGGG)_(n) repeated sequence. Based on extensive work in vivo aswell as in vitro, these are understood to include sunless tanning andrelated photoprotection, enhanced DNA repair capacity, cancer preventionand treatment, and immunomodulation.

Under normal conditions, the 3′ telomere overhang DNA sequence isbelieved to be folded back and concealed in a loop structure stabilizedby TRF2 [Griffith, J. D., et al., (1999), Cell 97, 503-514]. However,this sequence might be exposed if the telomere were distorted, forexample by ultraviolet (U.V.)-induced thymine dimers or carcinogenadducts involving guanine residues (as with cisplatin or benzo[a]pyrene)that could render the loop-back configuration unstable. Exposure of theTTAGGG (SEQ ID NO:11) repeat sequence could be the initial signalleading to a variety of DNA damage responses, dependent on cell type aswell as intensity and/or duration of the signal. These responses includecell cycle arrest, apoptosis, and a more differentiated sometimesadaptive phenotype, for example, increased melanin production (tanning).

The proposed model predicts that inability to repair damage to telomericDNA would lead to exaggerated damage responses, such as p53 inductionand apoptosis, as has been reported for LJV-irradiated xerodermapigmentosum cells that cannot efficiently remove DNA photoproducts(Dumaz, N., et al., 1998, Carcinogenesis 19: 1701-1704). This model isfurther consistent with the recent finding that transgenic mice withsupra-normal p53 activity are highly resistant to tumors, yet ageprematurely (Tyner, S. D., et al., 2002, Nature 415: 45-523). A DNAdamage recognition mechanism might have evolved to contain predominantlythymidine and guanine bases. The TTAGGG_(n) repeat sequence is anexcellent target for DNA damage, as dithymidine sites most commonlyparticipate in formation of U.V. photoproducts (Setlow, R. B. and W. L.Carrier. 1966, J Mol Biol 17: 237-254) and guanine is both the principalsite of oxidative damage, forming 8-oxoguanine [Kasai, H. and S.Nishimura, 1991. “Oxidative Stress: Oxidants and Antioxidants,” pp.99-116 In H. Sies (ed.) (London, Academic Press, Ltd.)], as well as thebase to which most carcinogens form adducts [Fnedberg, E. C., et al.,1995, pp. 1-58 In E. C. Friedberg, G. C. Walker and W. Siede, eds.

The invention includes methods for treating a hyperproliferativedisorder in a mammal, in which the therapy includes administering to themammal an effective amount of a composition comprising one or moreoligonucleotides as described herein. These methods can be appliedespecially to human subjects. Hyperproliferative disorders can becharacterized by benign growth of cells beyond a normal range, and whichsometimes may result in a benign tumor or widespread epidermalthickening, as in psoriasis. Also among the various hyperproliferativedisorders to be treated by these methods are cancer as it is manifestedin various forms and arising in various cell types and organs of thebody, for example, cervical cancer, lymphoma, osteosarcoma, melanoma andother cancers arising in the skin, and leukemia. Also among the types ofcancer cells to which the therapies are directed include but are notlimited to breast, lung, liver, prostate, pancreatic, ovarian, bladder,uterine, colon, brain, esophagus, stomach, and thyroid.

The oligonucleotides can be administered in the methods of treatmentdescribed herein as a single type of oligonucleotide or in a combinationcomprising with one or more different oligonucleotides. Oligonucleotideswithout a 5′ phosphate can be used in any of the methods of therapy fortreatment, or for the reduction of incidence of a disease or disorderdescribed herein. However, oligonucleotides having a 5′ phosphate arepreferred, as it has been shown that the 5′ phosphate improves uptake ofthe oligonucleotide into cells. The oligonucleotides can be at least 2nucleotides in length, preferably 2-200 nucleotides, and more preferablyfrom 2 to 20 nucleotides in length. Oligonucleotides 5-11 nucleotidesare more preferred.

Other methods of treatment for hyperproliferative diseases includingcancer that are encompassed by the present invention include theadministration of one or more oligonucleotides of the present inventionin combination with one or more chemotherapeutic agents. Suchchemotherapeutic agents include, but are not limited to vincristine,doxorubicin, prednisone, cyclophosphamide, busulphan, cisplatin,methotrexate, melphelan, chlorambucal, ra-c bleomycin, etoposide,fluorouroul and mitomycin as well as anticancer monoclonal antibodiessuch as Rituxin.

Oligonucleotides are relatively short polynucleotides. Polynucleotidesare linear polymers of nucleotide monomers in which the nucleotides arelinked by phosphodiester bonds between the 3′ position of one nucleotideand the 5′ position of the adjacent nucleotide. Unless otherwiseindicated, the “oligonucleotides” of the invention as described hereinhave a phosphodiester backbone.

To enhance delivery through the skin, the oligonucleotides of theinvention may be modified so as to either mask or reduce their negativecharges or otherwise alter their chemical characteristics. This may beaccomplished, for example, by preparing ammonium salts of theoligonucleotides using readily available reagents and methods well knownin the art. Preferred ammonium salts of the oligonucleotides includetrimethyl-, triethyl-, tributyl-, tetramethyl-, tetraethyl-, andtetrabutyl-ammonium salts. Ammonium and other positively charged groupscan be covalently bonded to the oligonucleotide to facilitate itstransport across the stratum corneum, using an enzymatically degradablelinkage that releases the oligonucleotide upon arrival inside the cellsof the viable layers of the epidermis.

Another method for reducing or masking the negative charge of theoligonucleotides includes adding a polyoxyethylene spacer to the 5′phosphate groups of the oligonucleotides and/or the internal phosphatesof the oligonucleotides using methods and reagents well known in theart. This, in effect, adds a 6- or 12-carbon modifier (linker) to thephosphate that reduces the net negative charge by +1 and makes theoligonucleotides less hydrophilic. Further negative charge reduction isachieved by adding a phosphoroamidite to the end of the polyoxyethylenelinker, thereby providing an additional neutralizing positive charge.

The phosphodiester backbone of the oligonucleotides of the presentinvention can also be modified or synthesized to reduce the negativecharge. A preferred method involves the use of methyl phosphonic acids(or chiral methylphosphonates), whereby one of the negatively chargedoxygen atoms in the phosphate is replaced with a methyl group. Theseoligonucleotides are similar to oligonucleotides having phosphorothioatelinkages which comprise a sulfate instead of a methyl group and whichare also within the scope of the present invention.

The oligonucleotides of the present invention can also take the form ofpeptide nucleic acids (PNAs) in which the bases of the nucleotides areconnected to each other via a peptide backbone.

Other modifications of the oligonucleotides such as those described, forexample, in U.S. Pat. Nos. 6,537,973 and 6,506,735 (both of which areincorporated herein by reference for all of the oligonucleotidemodifications described therein) and others will be readily apparent tothose skilled in the art.

The oligonucleotides can also be “chimeric” oligonucleotides which aresynthesized to have a combination of two or more chemically distinctbackbone linkages, one being phosphodiester. In one embodiment arechimeric oligonucleotides with one or more phosphodiester linkages atthe 3′ end. In another embodiment are chimeric oligonucleotides with oneor more phosphodiester linkages at the 3′ and 5′ ends.

As reported in Example 46 of PCT/US03/11393, 11-mer oligonucleotideswith sequence SEQ ID NO:5 were synthesized to contain phosphodiesterlinkages throughout, phosphorothioate linkages throughout, or acombination of linkages. One oligonucleotide had two phosphorothioatelinkages at the 5′ end; another oligonucleotide had two phosphorothioatelinkages at the 3′ end; still another had two phosphorothioate linkagesat each end. Oligonucleotides with phosphodiester linkages at the 3′ endwere found to be the most effective at stimulating reactions associatedwith senescence in fibroblasts. Thus, enzymatic cleavage at the 3′ endof the oligonucleotide maybe a step in induction of the senescenceresponse.

Sequence identity is determined by a best fit alignment of theoligonucleotide in question with (TTAGGG)_(n). The sequences arecompared at each position, and a determination of “match” or “no match”is made at each nucleotide position, and the percent of matches, withoutresorting to deletion or insertion in either sequence, is the percentidentity of the sequences as counted along the oligonucleotides inquestion. By illustration, GTTAGGG shares 100% sequence identity with(TTAGGG)_(n). pTT shares 100% sequence identity with (TTAGGG)_(n). (SeeTable 2 herein for additional examples of % identity)

Another part of the invention is a method for promoting differentiationof malignant cells in a mammal, the method comprising administering tothe mammal an effective amount of a composition comprising one or moreoligonucleotides which has at least 33% but less than 100% nucleotidesequence identity with (TTAGGG)_(n). A differentiated state, can in manyways, be considered the opposite of a malignant state. Depending on thecell type, differentiation can involve the regulation of expression of anumber of different genes to result in an increase or decrease incertain enzymatic activities, or cell surface proteins, for example. Forexample, melanocytes respond to oligonucleotides with an increase intyrosinase expression.

In PCT/US03/11393, an association was reported between the inhibition ofgrowth of cancer cells, caused by the cells taking up oligonucleotideswith sequence identity to the telomere repeat sequence, and an increasein the appearance on the cell surface of antigens typical ofdifferentiated cells, rather than cancer cells. Thus, a further methodof the invention is to enhance the expression of one or more surfaceantigens indicative of differentiation of cancer cells in a mammal, saidmethod comprising administering to the mammal an effective amount of oneor more oligonucleotides as described herein, for example, one or moreoligonucleotides which share at least 50% nucleotide sequence identitywith the vertebrate telomere overhang repeat.

This inducement of the cells to a more differentiated state, or to takeon one or more characteristics of differentiation, can be exploited inimmunotherapy methods. The surface antigens associated with adifferentiated state, fragments thereof, or synthetic peptides derivedfrom the studies of the externally exposed loops of the surfaceantigens, can be incorporated into a vaccine to induce a cancer patientto produce cytotoxic T lymphocytes against the cells displaying the cellsurface antigen. For example, in melanoma cells, the cell surfaceantigens MART-1, tyrosinase, TRP-1 or gp-100, or combinations thereof,can be made to increase on the cell surface when the cells take upoligonucleotides sharing at least 50% nucleotide sequence identity withthe telomere overhang repeat. See Example 29 of PCT/US03/11393. Thesecell surface antigens can become targets for immunotherapy, for exampleby vaccinations with the isolated cell surface antigen or peptideshaving amino acid sequences derived from the surface loops of theantigens. See, for example, Jäger, E. et al., Int. J. Cancer 66:470-476,1996; Kawakami, Y. et al., J. Immunol. 154:3961-3968, 1995; and deVries, T. J. et al., J. Pathol. 193:13-20, 2001.

Telomerase has been a target for antiproliferative methods based ontheories of using antisense oligonucleotides to bind to the RNA portionof the enzyme. However, the therapeutic methods described herein can beused independently of the presence or function of telomerase in thetarget cells. The telomere repeat overhang homolog pGTTAGGGTTAG (SEQ IDNO:5) was seen to bring about S-phase cell cycle arrest in normalfibroblasts and in cells of the osteosarcoma cell line Saos-2, neitherof which have telomerase activity. See Examples 32 and 34 ofPCT/US03/11393. Thus, for cancer cells, most of which have telomeraseactivity, but some of which do not, the present method can be usedregardless of telomerase activity. The inhibition of growth of thecancer cells is characterized by cell cycle arrest, apoptosis, and/ordifferentiation to a more differentiated state. A method for inhibitingthe growth of cancer cells in a mammal (e.g., human), operationallyindependent of the telomerase (+) or telomerase (−) state of the cancercells, is to administer to the mammal an effective amount of acomposition comprising one or more oligonucleotides which share at least50% nucleotide sequence identity with the telomere overhang repeat.

The experiments described in Example 34 of PCT/US03/11393 demonstratethat the function of a wild type p53 protein is also not necessary tobring about the S-phase cell cycle arrest in tumors or tumor cellstreated with an oligonucleotide with at least 50% sequence identity tothe telomere repeat sequence. A p53-null osteosarcoma cell line wasshown to respond to the addition of pGTTAGGGTTAG (SEQ ID NO:5) byarresting in S-phase. Thus, the method for inhibiting the growth ofcancer cells in a mammal (e.g., human), the method includingadministering to the mammal an effective amount of a compositioncomprising one or more oligonucleotides which share at least 50%nucleotide sequence identity with the telomere overhang repeat, can becarried out whether or not the target cells have normal p53 function.

The invention further comprises a method for preventing the sequelae ofexposure of the skin of a mammal to ultraviolet light—spongiosis,blistering or dyskeratosis, or any combination of these—by administeringto the skin an effective amount of a composition comprising one or moreoligonucleotides which share at least 50% nucleotide sequence identitywith the telomere overhang repeat. The steps or steps of this method canalso be used in the reduction in the incidence of skin cancer in ahuman, and is particularly applicable to reduce the occurrence of skincancer in patients with xeroderma pigmentosum or other geneticpredisposition to skin cancer. The method of applying to the skin aneffective amount of a composition comprising one or moreoligonucleotides which share at least 50% nucleotide sequence identitywith the telomere overhang repeat is also a method for enhancing repairof ultraviolet irradiation-induced damage to skin.

Oxidative damage is characterized by the reaction products of reactionsof molecules found in the cells with reactive oxygen species (ROS), suchas hydrogen peroxide, hydroxyl radicals, and superoxide. Oxidativedamage can result, for instance, from normal cellular metabolism, U.V.Airradiation, ionizing radiation, or exposure to a variety of chemicals.Reactive oxygen species can be measured in a number of ways. One assayemploys a probe dichlorofluorescein diacetate (Molecular Probes, Inc.),a colorless reagent that is taken up by the cells and becomesfluorescent upon oxidation by ROS. The level of fluorescence correlateswith the intracellular ROS level.

Applicants also described a method for reducing oxidative damage in amammal, the method comprising administering to the mammal, especially tothe skin of the mammal, an effective amount of a composition comprisingone or more oligonucleotides which share at least 50% nucleotidesequence identity with the human telomere overhang repeat. Preferred areembodiments in which the oligonucleotide is pGAGTATGAG (SEQ ID NO:1).See results in Examples 15, 21, 22, 23 and 52 of PCT/US03/11393suggesting that oligonucleotide treatment enhances the ability of cellsto repair oxidative DNA damage.

Applicants have further described a method for treating melanoma in amammal, comprising administering to the human an effective amount of acomposition comprising one or more oligonucleotides that share at least50% nucleotide sequence identity with the human telomere overhangrepeat. In particular cases, the oligonucleotide can be pGTTAGGGTTAG(SEQ ID NO:5); pTT can also be used in the method. Applicants have shownthe effectiveness of oligonucleotide therapy using human melanoma cellsin a mouse model. See PCT/US03/11393 Examples 30, 49, 50 and 51 andExample 9 set out below.

Another aspect of the invention concerns a method for reducingproliferation of keratinocytes in the skin of a human, in which themethod comprises applying to the skin an effective amount of acomposition comprising one or more oligonucleotides that share at least50% nucleotide sequence identity with the human telomere overhangrepeat. The method is applicable to disorders of the skin characterizedby proliferation of keratinocytes in the skin, such as seborrheickeratosis, actinic keratosis, Bowen's disease, squamous cell carcinomaor basal cell carcinoma. pTT is effective in the method.

The present invention includes the method of treating a disease ordisorder in a mammal, wherein the disease or disorder is characterizedby abnormal proliferation of cells, including, but not limited to, solidtumors, blood-cell related tumors (e.g., leukemias), tumor metastases,benign tumors (e.g., hemangiomas, acoustic neuromas, neurofibromas,trachomas, and pyogenic granulomas), including those in which cells areimmortalized such as apudoma, choristoma, branchioma, malignantcarcinoid syndrome, carcinoid heart disease, carcinoma, (e.g., Walker,basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, in situ,Krebs 2, merkel cell, mucinous, non-small cell lung, oat cell,papillary, schirrhous, bronchiolar, bronchogenic, squamous cell, andtransitional cell), histiocytic disorders, leukemia (e.g., b-cell, mixedcell, null-cell, T-cell, T-cell chronic, HTLV-II-associated, lyphocyticacute, lymphocytic chronic, mast-cell, and myeloid), histiocytosismalignant, Hodgkin's disease, immunoproliferative small, non-Hodgkin'slymphoma, plasmacytoma, reticuloendotheliosis, melanoma,chondroblastoma, chondroma, chondrosarcoma, fibroma, fibrosarcoma, giantcell tumors, hystiocytoma, lipoma, liposacaroma, mesothelioma, myxoma,myxosarcoma, osteoma, osteosarcoma, Ewing's sarcoma, synovioma,adenofibroma, adenolymphoma, carcinosarcoma, chordoma, mesenchymoma,mesonephroma, myosarcoma, ameloblatoma, cementoma, odontoma, teratoma,thymoma, throphoblastic tumor, adenocarinoma, adenoma, cholangioma,cholesteatoma, cyldindroma, cystadenocarcinoma, cystadenoma, garnulosacell tumor, gynandroblastoma, hepatoma, hidradenoma, islet cell tumor,leydig cell tumor, papilloma, sertoli cell tumor, theca cell tumor,leiomyoma, leiomyosarcoma, myoblastoma, myoma, myoscarcoma, rhabdomyoma,rhabdomyosarcoma, ependymoma, neuroblastoma, neuroepithelioma,neurofirbroma, neuroma, paraganglioma, paraganglioma nonchromaffin,antiokeratoma, angiolymphoid hyperplasia with eosinophilia, angiomasclerosing, angiomatosis, glomangioma, hemangioendothelioma, hemangioma,hemangiopericytoma, hemangiosarcoma, lymphangioma, lymphangiomyoma,lymphangiosarcoma, pinealoma, carcinoscarcoma, chondrosarcoma,cytosarcoma phyllodes, fibrosarcoma, hemangiosarcoma, leiomyosarcoma,leukosarcoma, liposcarcoma, lymphangiosarcoma, myoscarcoma, myxosarcoma,ovarian carcinoma, rhabdomyosarcoma, sarcoma (e.g., Ewing'sexperimental, Kaposi's, and mast-cell), neoplasms (e.g., bone, breast,digestive system, colorectal liver, pancreastic, pituitary, testicular,orbital, head and neck, central nervous system acoustic, pelvic,respiratory tract, and urogenital), neurofibromatosis, and cervicaldysplasia), and for treatment of other conditions in which cells haveincreased proliferation. Hyperproliferative disorders can also be thosecharacterized by excessive or abnormal stimulation of fibroblasts, suchas scleroderma, and hypertrophic scars (i.e., keloids).

The oligonucleotide or oligonucleotides to be used in therapies toalleviate hyperproliferative disorders such as cancer can be used in acomposition in combination with a pharmaceutically or physiologicallyacceptable carrier. Such a composition may also contain in addition,diluents, fillers, salts, buffers, stabilizers, solubilizers, and othermaterials well known in the art. Cationic lipids such as DOTAP[N-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium salts may be usedwith oligonucleotides to enhance stability. Oligonucleotides may becomplexed with PLGA/PLA copolymers, chitosan or fumaric acid/sebacicacid copolymers for improved bioavailability {where PLGA is[poly(lactide-co-glycolide)]; PLA is poly(L-lactide)}. The terms“pharmaceutically acceptable” and “physiologically acceptable” mean anon-toxic material that does not interfere with the effectiveness of thebiological activity of the active ingredient(s). The characteristics ofthe carrier will depend on the route of administration.

A composition to be used as an antiproliferative agent may furthercontain other agents which either enhance the activity of theoligonucleotide(s) or complement its activity or use in treatment, suchas chemotherapeutic or radioactive agents. Such additional factorsand/or agents may be included in the composition to produce asynergistic effect with the oligonucleotide(s), or to minimize sideeffects. Additionally, administration of the composition of the presentinvention may be administered concurrently with other therapies, e.g.,administered in conjunction with a chemotherapy or radiation therapyregimen.

The oligonucleotides as described herein can be used in combination withother compositions and procedures for the treatment of diseases. Forexample, a tumor may be treated conventionally with surgery, radiation,chemotherapy, or immunotherapy, combined with oligonucleotide therapy,and then oligonucleotides may be subsequently administered to thepatient to extend the dormancy of micrometastases and to stabilize andinhibit the growth of any residual primary tumor.

The compositions of the present invention can be in the form of aliposome in which oligonucleotide(s) of the present invention arecombined, in addition to other pharmaceutically acceptable carriers,with amphipathic agents such as lipids which exist in aggregated form asmicelles, insoluble monolayers, liquid crystals, or lamellar layers inaqueous solution. Suitable lipids for liposomal formulation include,without limitation, monoglycerides, diglycerides, sulfatides,lysolecithin, phospholipids, saponin, bile acids, and the like.

Pharmaceutical compositions can be made containing oligonucleotides tobe used in antiproliferative therapy. Administration of suchpharmaceutical compositions can be carried out in a variety ofconventional ways known to those of ordinary skill in the art, such asoral ingestion, inhalation, for example, of an aerosol, topical ortransdermal application, or intracranial, intracerebroventricular,intracerebral, intravaginal, intrauterine, oral, rectal or parenteral(e.g., intravenous, intraspinal, subcutaneous or intramuscular) route,or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenousinjection. The route of administration can be determined according tothe site of the tumor, growth or lesion to be targeted.

To deliver a composition comprising an effective amount of one or moreoligonucleotides to the site of a growth or tumor, direct injection intothe site can be used. Alternatively, for accessible mucosal sites,ballistic delivery, by coating the oligonucleotides onto beads ofmicrometer diameter, or by intraoral jet injection device, can be used.

Viral vectors for the delivery of DNA in gene therapy have been thesubject of investigation for a number of years. Retrovirus, adenovirus,adenoassociated virus, vaccinia virus and plant-specific viruses can beused as systems to package and deliver oligonucleotides for thetreatment of cancer or other growths. Adeno-associated virus vectorshave been developed that cannot replicate, but retain the ability toinfect cells. An advantage is low immunogenicity, allowing repeatedadministration. Delivery systems have been reviewed, for example, inPage, D. T. and S. Cudmore, Drug Discovery Today 6:92-1010, 2001.

Studies carried out using oligonucleotides on the theory of theirinhibiting the function of a target nucleic acid (antisenseoligonucleotides), most of these studies carried out withphosphorothioate oligonucleotides, have found effective methods ofdelivery to target cells. Antisense oligonucleotides in clinical trialshave been administered in saline solutions without special deliveryvehicles (reviewed in Hogrefe, R. I., Antisense and Nucleic Acid DrugDevelopment 9:351-357, 1999).

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the fluids of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example, sealed ampules and vials, and may be stored ina freeze-dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example, water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described. A preferred pharmaceutical compositionfor intravenous, cutaneous, or subcutaneous injection should contain, inaddition to oligonucleotide(s) of the present invention, an isotonicvehicle such as Sodium Chloride Injection, Ringer's Injection, DextroseInjection, Dextrose and Sodium Chloride Injection, Lactated Ringer'sInjection, or other vehicle as known in the art. The pharmaceuticalcomposition of the present invention may also contain stabilizers,preservatives, buffers, antioxidants, or other additives known to thoseof skill in the art.

Use of timed release or sustained release delivery systems are alsoincluded in the invention. Such systems are highly desirable insituations where surgery is difficult or impossible, e.g., patientsdebilitated by age or the disease course itself, or where therisk-benefit analysis dictates control over cure. One method is to usean implantable pump to deliver measured doses of the formulation over aperiod of time, for example, at the site of a tumor.

A sustained-release matrix can be used as a method of delivery of apharmaceutical composition comprising oligonucleotides, especially forlocal treatment of a growth or tumor. It is a matrix made of materials,usually polymers, which are degradable by enzymatic or acid/basehydrolysis or by dissolution. Once inserted into the body, the matrix isacted upon by enzymes and body fluids. The sustained-release matrixdesirably is chosen from biocompatible materials such as liposomes,polylactides (polylactic acid), polyglycolide (polymer of glycolicacid), polylactide co-glycolide (co-polymers of lactic acid and glycolicacid) polyanhydrides, poly(ortho)esters, polyproteins, hyaluronic acid,collagen, chondroitin sulfate, carboxylic acids, fatty acids,phospholipids, polysaccharides, nucleic acids, polyamino acids, aminoacids such as phenylalanine, tyrosine, isoleucine, polynucleotides,polyvinyl propylene, polyvinylpyrrolidone and silicone. A preferredbiodegradable matrix is a matrix of one of either polylactide,polyglycolide, or polylactide co-glycolide (co-polymers of lactic acidand glycolic acid).

Also encompassed by the present invention is combination therapies inwhich one or more oligonucleotides of the present invention areadministered in combination with other therapies. For example, patientsmay receive suboptional doses of both an oligonucleotide of the presentinvention along with suboptional doses of a chemotherapeutic agent suchas vincristin or doxyrubicin—the combination of which has been shown toinduce apoptosis in malignant B-cells.

The amount of oligonucleotide of the present invention in thepharmaceutical composition of the present invention will depend upon thenature and severity of the condition being treated, and on the nature ofprior treatments which the patient has undergone. For a human patient,the attending physician will decide the dose of oligonucleotide of thepresent invention with which to treat each individual patient.Initially, the attending physician can administer low doses and observethe patient's response. Larger doses may be administered until theoptimal therapeutic effect is obtained for the patient, and at thatpoint the dosage is not increased further. The duration of therapy usingthe pharmaceutical composition of the present invention will vary,depending on the severity of the disease being treated and the conditionand potential idiosyncratic response of each individual patient.

It is apparent from the data disclosed herein including data inapplications and patents incorporated herein by reference that theoligonucleotides of the present invention have pleiotropic effects ongene expression in tumor cells (e.g., MART-1, tyrosinase, TRP-1 orgp-100 and others). Based on these pleiotropic effects in cancer cells,the oligonucleotides of the present invention can be said to induce inthose cells an “anticancer expression profile” all or some of which maybe further exploited as possible targets for anti-cancer intervention.

The invention is further illustrated by the following non-limitingExamples.

EXAMPLES Example 1

Based on our observation that the oligonucleotides of the presentinvention have the same relative molar efficacy in causing pigmentationin melanocytes, apoptosis and cellular senescence in malignant cells, aretrospective comparison of activities of a number of oligonucleotideswas made and the relative molar efficacy of the oligonucleotides withrespect to one another was calculated with the results set out inTable 1. Based on these results, we have concluded that the followingparameters are among those that are important in determining efficacy ofthe oligonucleotides of the present invention.

-   -   1) percent tolemere identity;    -   2) number of hydrolyzable linkages;    -   3) guanine content;    -   4) sequence motif        The comparisons also reveal that the presence of cytosine        residues in the oligonucleotides can have a negative impact on        molar efficiency.

TABLE 1 Relative Molar Efficacy of Tested T-Oligos Sequence Activity T0 * TT 1 AA 0 TTA 1.5 TTAG 2 * GAGTATGAG (SEQ ID NO: 37) 3  AGTATGA 2  GTATG 1.5   CATAC 0 GCATGCATGCATTACGTACG (SEQ ID NO: 38) 0 GTTAGGGTTAG(SEQ ID NO: 2) 6 CTAACCCTAAC (SEQ ID NO: 3) 0 GTACGTACGTA (SEQ ID NO: 4)0 TTAGGG (SEQ ID NO: 6) 3 TTCGGG (SEQ ID NO: 7) 0 CTAGGG (SEQ ID NO: 8)0.5 TTAGGC (SEQ ID NO: 9) 0.5 GGTAGGTGTAGGATT (SEQ ID NO: 10) 8GGTAGGTGTAGGTTA (SEQ ID NO: 11) 7 GGTTAGGTGTAGGTT (SEQ ID NO: 12) 7GGTTAGGTGGAGGTTT (SEQ ID NO: 13) 8 GGTTAGGTGTAGGTTT (SEQ ID NO: 14) 10GGTTAGGTTAGGTTA (SEQ ID NO: 15) 7 GTTAGGGTTAG (SEQ ID NO: 5) 6GGTAGGTGTAGGGTG (SEQ ID NO: 16) 9  GTTAGGGTT (SEQ ID NO: 17) 6 TTAGGGTTA (SEQ ID NO: 18) 4 GTTAGGTTTAAGGTT (SEQ ID NO: 19) 6GGTCGGTGTCGGGTG (SEQ ID NO: 20) 1 GGCAGGCGCAGGGCG (SEQ ID NO: 21) 1GTTAGGGTTAGGGTT (SEQ ID NO: 22) 8     GGGTTAGGG (SEQ ID NO: 23) 7G_(S)T_(S)T_(S)A_(S)G_(S)G_(S)G_(S)T_(S)T_(S)A_(S)G (SEQ ID NO: 24) 0G_(S)T_(S)TAGGGTT_(S)A_(S)G (SEQ ID NO: 25) 1 GTTAGGGTT_(S)A_(S)G (SEQID NO: 26) 2 G_(S)T_(S)TAGGGTTAG (SEQ ID NO: 27) 3 Note: All sequenceshave a 5′ phosphate group, and all have phosphodiester linkage unlessotherwise indicated: x_(s)x = phosphorothioate linkage. * Tested withoutthe 5′ phosphate group: no activity (no uptake).

Example 2 Sequence Parameters

In order to further examine the effect of various nucleotide sequenceparameters on the relative efficacy of T-oligos to induce apoptosis inMM-AN melanoma several oligonucleotides were synthesized which hadvarying % identity with the telomere repeat and which were varied withrespect to the number of bases, number of guanine bases, number of GT's,number of GGT's, and the number of GGTTs. Apoptosis studies wereundertaken as described in PCT/US03/11393 incorporated herein byreference. (See e.g. Examples 13, 28) MM-An melanoma cells were exposedto each of the T-oligos shown in Table 3 at a concentration of 40 μM for96 hours. After 96 hours the cells were stained with propidium iodideand the percent of DNA content in the sub GI portion of the cell cyclewas determined by FACS analysis. Relative activity was determined bycomparison to the relative activities shown in Table 1. The sequenceparameters that were investigated include: % identity with the telomererepeat sequence (TTAGGG)_(n); the number of bases, the number of guanineresidues and the presence or absence of various sequence motifsincluding the number of GTs, GGTs, and GGTTs.

The most active T-oligo in this study was shown to be GGTTGGTTGGTTGGTT,(SEQ ID NO:29) which was 56% identical to the (TTAGGG)_(n), telomererepeat has 16 bases 8 guanine residues, four GT sequences, four GGTs andfour GGTTS and which showed relative activity of 12. Typically,GGTTGGTTGGTTGGTT (SEQ ID NO:29) had relative activity of 12 yielding 61%apoptotic cells compared to 43%-47% apoptotic cells seen witholigonucleotides having a relative activity of 10 while GTTAGGGTTAG (SEQID NO:5) with a relative activity of 6 yielded 25% apoptotic cells inthe assay. Notably, the sequence GGAGGAGGAGGAGGA (SEQ ID NO:31) whichwas 47% identical to the telomere repeat has 10 G residues but whichlacked any GT, GGT, or GGTT repeats had no activity in this assay. Ofadditional note is the fact that the T-oligo TGTGGTTGTGGTGTGG (SEQ IDNO:33) which is 15 bases in length, only 40% identical to the telomererepeat, has 9 Gs, 5GTs, and 2 GGTTs has a relative molar efficacy of 10.The T-oligo TAGTGTTAGGTGTAG (SEQ ID NO:34) which is 15 bases in lengthonly 40% identical to the telomere repeat has 6 Gs 4 GTs and 1 GGT andhas a relative molar efficacy of 10.

TABLE 2 Relative % Sequence Activity Identity # Bases #G #GT #GGT #GGTTGTTAGGGTTAG 6 100 11 5 2 1 1 (SEQ ID NO: 5) GGTTAGGGTGTAGGTTT 10 81 16 74 3 2 (SEQ ID NO: 28) GGTTGGTTGGTTGGTT 12 56 16 8 4 4 4 (SEQ ID NO: 29)GGTGGTGGTGGTGGT 10 60 15 10 4 4 0 (SEQ ID NO: 30) GGAGGAGGAGGAGGA 0 4715 10 0 0 0 (SEQ ID NO: 31) GGTGTGGTGTGGTGT 10 60 15 9 6 3 0 (SEQ ID NO:32) TGTGGTGTGGTGTGG 10 40 15 9 5 2 0 (SEQ ID NO: 33) TAGTGTTAGGTGTAG 940 15 6 4 1 0 (SEQ ID NO: 34)

Example 3 Dose-Response Effects of T-Oligos on Apoptosis in MM-AN Cells

Experiments were conducted comparing the ability of different T-oligosat a range of concentrations to induce apoptosis in MM-AN melanomacells. Apoptosis studies were undertaken as described in PCT/US03/11393(See e.g. Examples 13 and 28) and treated once in triplicate with eachT-oligo at each dose (1, 5 and 10 μM) and subjected to FACS analysisafter 96 hours as described in PCT/US03/11393. The T-oligos used werepGTTAGGGTTAG (SEQ ID NO:5), p(GGTT)₄ (SEQ ID NO:29) and p(GGTT)₅ (SEQ IDNO:35). Results are shown in FIGS. 1 and 4. The results are compatiblewith earlier experiments and show that the standard (GTTAGGGTTAG) (SEQID NO:5) is less effective at inducing apoptosis than either thep(GGTT)₄ (SEQ ID NO:29) or p(GGTT)₅ (SEQ ID NO: 35), which werecomparable in efficacy. In a separate experiment (GGTT)₄ (SEQ ID NO:29)with and without 5′ phosphorylation were equally active in the MM-ANcell assay for apoptosis. Maximally effective doses for both thep(GGTT)₄ (SEQ ID NO:29) and p(GGTT)₅, (SEQ ID NO:35) as determined inother experiments was determined to be 20 μM.

Example 4 Apoptosis and H2AX Phosphorylation

Experiments were conducted to examine the ability of variousoligonucleotides, to induce apoptosis in MM-AN cells and to determinetheir effects on phosphorylation of histone H2AX in MCF7 cells, a breastcancer cell line. The apoptosis data was obtained using methods set andin Examples 13 and 28 of PCT/US03/11393 are from a FACS analysis (%cells with <2N DNA content) in duplicate dishes with moderatevariability. Data is shown in FIG. 2. These data indicate that all ofthe tested oligonucleotides are comparably active to the previouslytested 15-mer (SEQ ID NO:34) and more active than the standard 11-mer(SEQ ID NO:5).

H2AX phosphorylation results are shown in FIG. 3 and indicate that theoligonucleotides are capable of inducing phosphorylation of H2AX inMCF-7 cells.

Example 5 The Effects of T-Oligos on Phosphorylation of H2AX and p53

Additional studies were conducted to assess the ability of certainT-oligos to phosphorylate the DNA damage response protein histone H2AXand p53 in human breast cancer cells. Cells were treated with theGTTAGGGTTAG (SEQ ID NO:5) at (40 μm), its complement or diluent andresulting phosphorylation of histone H2AX and p53 as measured by westernblot analysis and apoptosis of breast cancer cells was assayed. Theresults of this study indicate that (pGTTAGGGTTAG) (SEQ ID NO:5) versuscomplimentary control T-oligo or diluent alone strongly inducesphosphorylation of H2AX and p53 in human breast cancer (MCF-7) cells(see FIGS. 4 and 5) but only very weakly induces these phosphorylationsin normal human mammary epithelial cells. Similarly, GTTAGGGTTAG (SEQ IDNO:5) strongly induces apoptosis in MCF-7 and BT-20 breast carcinoma(measured as described above) cells but only very weakly inducesapoptosis in normal mammary epithelial cells (data not shown). A singlesupplementation of MCF-7 or BT-20 cells on day zero causes permanentgrowth arrest (at least through 14 or 8 days respectively), even whenthe cells are re-fed with serum containing fresh medium that lacks theoligonucleotide; while the same supplementation of normal mammaryepithelial cells on day zero arrests their growth through day 4, butupon re-feeding with fresh medium, the cells again begin to grow at adoubling rate comparable to that of the diluent treated control cells.(FIGS. 6 and 7)

Example 6 Effect of T-Oligos on Survival of Mice Injected with HumanBreast Cancer Cells

Animals were injected in the tail vein with MCF-7 cells at a dosagewhich results in death of all the injected mice within about 42 days.Mice were treated with diluent alone with 60 nmoles or 120 nmoles ofpGTTAGGGTTAG (SEQ ID NO:5) with pGGTTAGGTTTAGGTTT (SEQ ID NO:36) and 10μM and 20 μM, compared to diluent (saline) alone. The results are shownin FIG. 9 which shows that both T-oligos greatly prolong survival ofmice that have received tail vein injections of MCF-7 cells with the 16mer being more effective than the 11 mer.

Example 7 Effects of T-Oligos on Squamous Cell Carcinoma in Mice

XPA KO mice lacking the XPA DNA repair protein were irradiated twiceweekly with 6 mJ/cm² of U.V.B until squamous cells tumor developed.Tumors were then injected three times weekly with 40 μM, GTTAGGGTTAG(SEQ ID NO:5), 10-40 μl per injection depending on tumor size. Theresults of this study shown in FIG. 10 indicates that aggressivelygrowing squamous cell carcinoma can regress substantially or completelyas a result of treatment with GTTAGGGTTAG (SEQ ID NO:5).

Example 8 Effects of T-Oligos on Growth of MM-AN Melanoma Cells in MiceSCID Mice

Experiments were conducted to examine the effect of two T-oligos, an11mer (pGTTAGGGTTAG) (SEQ ID NO:5) and a 16-mer (pGGTTAGGTGTAGGTTT) (SEQID NO:14), compared to control animals. In the first experiment, 2×10⁶MM-AN cells were injected subcutaneously into the flank of each animaland on day 5, when tumors were first clinically apparent, the micereceived twice daily intravenous injections for 5 days (10 injections)of either the 11-mer (120 nMoles), 16-mer (60 nMoles), or no treatmentat all. Results are shown in FIG. 11. Both treatments were quiteeffective and the half-dose 16-mer was slightly but not statisticallysuperior to the full-dose 11-mer. In a second experiment 2×10⁶ MM-ANcells were injected into the tail vein, a procedure known to causewidespread metastases, and beginning on day 3 the animals were injectedtwice daily for 5 days with either the 11-mer or 16-mer or Hank'sbuffered saline solution (diluent alone) as a control. The experimentwas completed when the control group (CTL) began to lose weight andappear ill. The data show that both oligonucleotides were quiteeffective in reducing number and size of metastases, with the 16-mer athalf-dose again slightly more effective than the 11-mer at full-dose andtumor volume (See FIGS. 11-14).

Example 9 Oligonucleotide Killing of Non-Hodgkins Lymphoma Lymphoblasts

The mechanism by which oligonucleotides of the present invention affectcells is partially known. It is known that oligonucleotides activate theATM kinase, leading to modification of the p95/Nbs1 protein responsiblefor S-phase arrest of the cell cycle. In the presence of continuousmitogenic stimulation, a G₁/G₀ arrest is subsequently achieved,presumably through p53 and p21. Experiments were conducted to extendsuch studies to ascertain the effects of GTTAGGGTTAG (SEQ ID NO:2)(referred to hereinafter as T-oligo) on malignant B cell lines(DLCL—diffuse large B-cell lymphoma), wherein the cells were treatedwith 20 μM of the oligonucleotide followed by FACS analysis as describedabove.

Rather than a G₂/M arrest followed by apoptosis at 48 h, treatment ofall cells (40,000) with SEQ ID NO:2 (20 μM) caused a very early S phasearrest, which became a more pronounced by 72 h. All the cell linesbehaved with similar kinetics, and cell death was through caspase-3elevation and apoptosis as before. It is thought that the presentT-oligo induces an early S phase arrest largely through phosphorylationof the p95/Nbs1 protein. Apoptotic cells were not a significant fractionof the population until 48 h, when 41% apoptotic cells were observed.After 72 h, 67% of the treated cells were apoptotic. Stabilization ofp53 was observed in all cell types tested, but was transient in Toledocells and MOLT-4 cells, whereas stabilization was sustained in RL andFarage cells. Unlike doxorubicin, T-oligo induced p53 in Toledo cells,indicating that p53 induction was still possible here, but not inMOLT-4, as expected. Taken together with the cell cycle profiles, theevidence clearly supports a conclusion that the apoptotic pathways inthe doxorubicin treated cells and T-oligo cases are not the same. The Sphase arrest in T-oligo treated DLCL cells is consistent with the arrestseen in MM-AN melanoma cells, but it occurs earlier in DLCL cells;requiring up to 96 h in MM-AN cells and fibroblast-type adherent cells.

Cyclophilin loading controls confirmed equal loading for all cell types,including MOLT-4. A dose-response experiment shows that caspase-3induction at 4 h after T-oligo exposure (40,000 cells) did not appear tosaturate with increasing T-oligo concentration, unlike doxorubicintreated cells. Toledo cells were the most sensitive to T-oligotreatment, showing a steeper dose-response curve, and MOLT-4 the mostresistant, suggesting that lack of p53 expression is associated withresistance. It was in some sense surprising that Toledo cells were moresensitive than RL and Farage, because their p53 induction was brief andtransient. These differences were consistent with apoptotic cell countsat each time point in each case, although all cell types were killedcompletely by 96 h. Because oligonucleotides of this size withphysiologic phosphodiester linkage have a half-life in culture of 4-6 h,the effective exposure time of DLCL cells to T-oligos in theseexperiments is probably far less than the 72 h in vitro incubationperiod shown.

Example 10 Vincristine and T-Oligo Synergistic Killing of DLCL Cells

Three single agents in common use as part of the CHOP+R(cyclophosphamide, doxorubicin, vincristine, prednisone, Rituxin)(Godwin, et al., Clinical Lymphoma 2:155-163 (2001) standard of carewere tested, in combination with T-oligo described in Example 9:doxorubicin, anti-CD20 and vincristine. We chose doxorubicin because wehad already obtained interesting information on its behavior inpromoting apoptosis in DLCL cells; and combination with treatment withour oligonucleotide. A purified mouse IgG_(2b), κ monoclonal anti-humanCD20 (eBioscience) was used, which is similar to Rituximab, because allthe DLCL lines we used were CD20+ and might be susceptible toCD20-receptor mediated apoptosis. Monomeric Rituximab chemosensitizesdrug-resistant NHL cells through CD20 signaling, selectivelydownregulating anti-apoptotic factors, such as Bcl-2, although inpatients, there are two other major mechanisms of Rituximab action notseen in tissue culture experiments such as these: complement-mediatedcytotoxicity through the F_(c) portion of the chimeric molecule andantibody-dependent cellular cytotoxicity. We hypothesized that theapoptotic action of anti-CD20 alone might be sufficient to combine withT-oligo apoptotic action. Finally, vincristine was chosen because it toois a component of CHOP, and, like doxorubicin and Rituximab, is likelyto work through an apoptotic pathway independent of T-oligo. Vincristineand its Vinca alkaloid sister compound vinblastine are spindle poisonsand therefore act as mitotic blockers. In each case, the anticipatedmechanistic differences with T-oligo action were hypothesized to createadditive or synergistic effects in combination, given the firmlyestablished principle of cancer chemotherapy that multiple, independentmodes of drug action are always more effective than single agents oragents that act in the same pathway.

Submaximal concentrations of doxorubicin (50 pM) or anti-CD20 (1 μg/ml)and of T-oligo (2 μM) SEQ ID NO:2 were tested and assayed caspase-3induction (6000 cells) was assayed, we found that doxorubicin andanti-CD20 did not synergistically induce caspase-3 activity with theT-oligo. In the absence of additional control experiments, we cannotcomment further on these negative results. However, assay of caspase-3activity 12 h after addition of vincristine (25 nM; Sigma) and T-oligo(2 μM) together showed synergistic induction of caspase-3 activity,compared to either single agent. DNA synthesis was measured by BrdUincorporation as detected with FITC anti-BrdU antibody (BD) andcorrelated with DNA content as detected with 7-aminoactinomycin Dfluorescence of fixed cells by flow cytometry (FACS). Untreated controlToledo cells showed normal G_(O)/G₁ phase, S phase and G₂/M phasepopulations, such as would be expected for proliferating malignantcells, but after 12 hours' treatment with the drug combination werecompletely devoid of DNA. However, genomic DNA had not yet degradedsignificantly into the familiar apoptotic pattern, which was indeedobserved at later times and correlated with caspase-3 activity. For suchlow doses of T-oligo and vincristine, we found that caspase-3 inductionwas best measured at the later time point of 12 h, rather than at 4 hsuch as we had performed previously, because significant differenceswere not detectable at 4 h. Furthermore, at higher vincristineconcentrations (e.g., 0.25 μM), T-oligo effects were swamped, and again,no differences were detected, due to the high toxicity of vincristine.Observations of BrdU incorporation were consistent with this pattern. Itis possible that MOLT-4 cells might show less synergy, given their p53status, although we have not yet tested these cells. These resultssuggest that low dose vincristine and low dose T-oligo work welltogether with minimal side effects in DLCL patients.

Example 11 T-Oligo Causes Cell Cycle Arrest of Normal B Cells but notApoptosis

In order to establish a therapeutic window for the use of T-oligo,either in combination with vincristine or not, it is essential to studyits effect on normal cells. All anti-cancer chemotherapeutic drugs arehighly toxic and dosages must compromise between efficacy and toxicity.One of the unique reported features of T-oligo is that by mimicking theexposure of telomeric oligonucleotides, the drug activates the sensorresponsible for monitoring telomere structure, but the p53-dependentcell cycle arrest that follows is not then followed by apoptosis innormal fibroblasts, the system in which T-oligos (and the thymidinedinucleotides from which T-oligos were deduced) were originally studied.Cell cycle arrest is temporary and, presumably because of the lack ofactual DNA damage, cells eventually return to their normal metabolism.

EQUIVALENTS

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method of treating a hyperproliferative disorder in a mammal themethod comprising administering to the mammal an effective amount of acomposition comprising one or more oligonucleotides, saidoligonucleotide having between 2 and 200 bases and having at least 33%but less than 100% identity with the sequence (TTAGGG)_(n), andoptionally having a 5′ phosphate, and when said oligonucleotidecomprises the sequence 5′-RRRGGG-3′ (R=any nucleotide) saidoligonucleotide has a guanine content of 50% or less.
 2. The method ofclaim 1 wherein said oligonucleotide lacks cytosine.
 3. A method forinhibiting growth of cancer cells in a human comprising administering tothe human an effective amount of a composition comprising one or moreoligonucleotides, said oligonucleotide having between 2 and 200 basesand having at least 33% but less than 100% identity with the sequence(TTAGGG)_(n), and optionally having a 5′ phosphate, and when saidoligonucleotide comprises the sequence 5′-RRRGGG-3′ (R=any nucleotide)said oligonucleotide has a guanine content of 50% or less.
 4. The methodof claim 3 wherein said oligonucleotide lacks cytosine.
 5. A method ofpromoting differentiation of malignant cells in a mammal comprising oneor more oligonucleotides, said oligonucleotide having between 2 and 200bases and having at least 33% but less than 100% identity with thesequence (TTAGGG)_(n), and optionally having a 5′ phosphate, and whensaid oligonucleotide comprises the sequence 5′-RRRGGG-3′ (R=anynucleotide) said oligonucleotide has a guanine content of 50% or less.6. The method of claim 5 wherein said oligonucleotide lacks cytosine. 7.A method of inducing apoptosis in a cancer cell said method comprisingadministering to the mammal one or more oligonucleotides, saidoligonucleotide having between 2 and 200 bases and having at least 33%but less than 100% identity with the sequence (TTAGGG)_(n), andoptionally having a 5′ phosphate, and when said oligonucleotidecomprises the sequence 5′-RRRGGG-3′ (R=any nucleotide) saidoligonucleotide has a guanine content of 50% or less.
 8. The method ofclaim 7 wherein said oligonucleotide lacks cytosine.
 9. The method forreducing the occurrence of skin cancer in a human the method comprisingone or more oligonucleotides, said oligonucleotide having between 2 and200 bases and having at least 33% but less than 100% identity with thesequence (TTAGGG)_(n), and optionally having a 5′ phosphate, and whensaid oligonucleotide comprises the sequence 5′-RRRGGG-3′ (R=anynucleotide) said oligonucleotide has a guanine content of 50% or less.10. The method of claim 9 wherein said oligonucleotide lacks cytosine.